Recombinant P53 adenovirus methods and compositions

ABSTRACT

Described are simplified and efficient methods for preparing recombinant adenovirus using liposome-mediated cotransfection and the direct observation of a cytopathic effect (CPE) in the transfected cells. Also disclosed are compositions and methods involving novel p53 adenovirus constructs, including methods for restoring p53 function and tumor suppression in cells and animals having abnormal p53.

This application is a continuation of U.S. patent application Ser. No.08/145,826, filed on Oct. 29, 1993, which issued on Jun. 25, 2002 asU.S. Pat. No. 6,410,010.

The government owns rights in the present invention pursuant to NIHgrants RO1 CA 45187 and CA 16672.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the area of recombinanttechnology. In some aspects, it concerns simplified and efficientmethods of generating recombinant adenovirus. In other aspects, novelcompositions and methods involving p53 adenovirus constructs areprovided, including methods for restoring normal p53 functions andgrowth suppression to cells with abnormal p53.

2. Description of Related Art

Current treatment methods for cancer, including radiation therapy,surgery, and chemotherapy, are known to have limited effectiveness. Lungcancer alone kills more than 140,000 people annually in the UnitedStates. Recently, age-adjusted mortality from lung cancer has surpassedthat from breast cancer in women. Although implementation ofsmoking-reduction programs has decreased the prevalence of smoking, lungcancer mortality rates will remain high well into the 21st century. Therational development of new therapies for lung cancer will depend on anunderstanding of the biology of lung cancer at the molecular level.

It is now well established that a variety of cancers are caused, atleast in part, by genetic abnormalities that result in either the overexpression of one or more genes, or the expression of an abnormal ormutant gene or genes. For example, in many cases, the expression ofoncogenes is known to result in the development of cancer. “Oncogenes”are genetically altered genes whose mutated expression product somehowdisrupts normal cellular function or control (Spandidos et al., 1989).

Most oncogenes studied to date have been found to be “activated” as theresult of a mutation, often a point mutation, in the coding region of anormal cellular gene, i.e., a “proto-oncogene”, that results in aminoacid substitutions in the expressed protein product. This alteredexpression product exhibits an abnormal biological function that takespart in the neoplastic process (Travali et al., 1990). The underlyingmutations can arise by various means, such as by chemical mutagenesis orionizing radiation. A number of oncogenes and oncogene families,including ras, myc, neu, raf, erb, src, fms, jun and abl, have now beenidentified and characterized to varying degrees (Travali et al., 1990;Bishop, 1987).

During normal cell growth, it is thought that growth-promotingproto-oncogenes are counterbalanced by growth-constraining tumorsuppressor genes. Several factors may contribute to an imbalance inthese two forces, leading to the neoplastic state. One such factor ismutations in tumor suppressor genes (Weinberg, 1991).

An important tumor suppressor gene is the gene encoding the cellularprotein, p53, which is a 53 kD nuclear phosphoprotein that controls cellproliferation. Mutations to the p53 gene and allele loss on chromosome17p, where this gene is located, are among the most frequent alterationsidentified in human malignancies. The p53 protein is highly conservedthrough evolution and is expressed in most normal tissues. Wild-type p53has been shown to be involved in control of the cell cycle (Mercer,1992), transcriptional regulation (Fields et al., 1990, and Mietz etal., 1992), DNA replication (Wilcock and Lane, 1991, and Bargonetti etal., 1991), and induction of apoptosis (Yonish-Rouach et al., 1991, and,Shaw et al., 1992).

Various mutant p53 alleles are known in which a single base substitutionresults in the synthesis of proteins that have quite different growthregulatory properties and, ultimately, lead to malignancies (Hollsteinet al., 1991). In fact, the p53 gene has been found to be the mostfrequently mutated gene in common human cancers (Hollstein et al., 1991;Weinberg, 1991), and is particularly associated with those cancerslinked to cigarette smoke (Hollstein et al., 1991; Zakut-Houri et al.,1985). The overexpression of p53 in breast tumors has also beendocumented (Casey et al., 1991).

One of the most challenging aspects of gene therapy for cancer relatesto utilization of tumor suppressor genes, such as p53. It has beenreported that transfection of wild-type p53 into certain types of breastand lung cancer cells can restore growth suppression control in celllines (Casey et al., 1991; Takahasi et al., 1992). Although DNAtransfection is not a viable means for introducing DNA into patients'cells, these results serve to demonstrate that supplying wild type p53to cancer cells having a mutated p53 gene may be an effective treatmentmethod if an improved means for delivering the p53 gene could bedeveloped.

Gene delivery systems applicable to gene therapy for tumor suppressionare currently being investigated and developed. Virus-based genetransfer vehicles are of particular interest because of the efficiencyof viruses in infecting actual living cells, a process in which theviral genetic material itself is transferred. Some progress has beenmade in this regard as, for example, in the generation of retroviralvectors engineered to deliver a variety of genes. However, majorproblems are associated with using retroviral vectors for gene therapysince their infectivity depends on the availability of retroviralreceptors on the target cells, they are difficult to concentrate andpurify, and they only integrate efficiently into replicating cells.

Adenovirus vector systems have recently been proposed for use in certaingene transfer protocols, however, the current methods for preparingrecombinant adenovirus have several drawbacks. These methods rely oncalcium phosphate-mediated transfection of expression vectors andadenoviral plasmids into host cells and subsequent plaque assays on thetransfected cells. These types of transfection steps and assays areinefficient and typically result in low levels of viral propagation.

There remains, therefore, a clear need for the development of newmethods for introducing tumor suppressor genes, such as p53, into cellsas a means for restoring growth suppression. Methods for producingrecombinant adenovirus which avoid calcium-phosphate mediatedtransfection and agarose overlay for plaque assays would also beadvantageous.

SUMMARY OF THE INVENTION

The present invention addresses the foregoing and other problems byproviding efficient methods for producing recombinant adenovirus, suchas p53 adenovirus, and effective means by which to restore p53 functionsto cells with aberrant p53. Recombinant adenovirus vectors and virionsare disclosed, as are methods of using such compositions to promote wildtype p53 expression in cells with aberrant p53 functions, such as cancercells. Also disclosed is a simplified protocol for propagatingrecombinant adenovirus using liposome-mediated DNA transfection followedby observation of cytopathic effect (CPE) and, preferably, polymerasechain reaction (PCR) analysis.

Furthermore, utilizing this new method for generating and propagatingrecombinant adenoviruses, it is envisaged that other genes may beincorporated into the virion genome. These genes could include tumorsuppressor genes such as the retinoblastoma (rb) gene, antisenseoncogenes, i.e. anti-c-myc and anti-k-ras, and other growth controlrelated genes for cancer gene therapy.

Using the present invention the inventors have demonstrated a remarkableeffect in controlling metastatic growth. The Ad5CMV-p53 recombinantadenovirus was shown to markedly reduce the growth rate of transformedcells. The virus inhibited tumorigenicity of virus-infected H358 cells.Furthermore it prevented orthotopic lung cancer growth when the viruswas instilled intratracheally following the intratracheal inoculation ofthe H226Br cells. The inhibition of tumorigenicity also suggests thateven transient expression of a high-level of the p53 protein may beenough to induce a tumoricidal effect.

In one specific embodiment, this invention concerns vector constructsfor introducing wild type p53 genes into target cells, such as targetcells suspected of having mutant or aberrant p53 genes, includingmalignant cell types. These embodiments involve the preparation of agene expression or transcription unit wherein the p53 gene is placedunder the control of a promoter and the unit is incorporated into anadenoviral vector within a recombinant adenovirus. The invention as awhole is surprising and advantageous for several reasons. Firstly, itwas previously thought that p53 virus could not be generated into apackaging cell, such as those used to prepare adenovirus, as it would betoxic; secondly, E1B of adenovirus binds to p53 and thus interferes withits function; thirdly, once generated, the p53 adenovirus was found tobe unexpectedly effective at inhibiting the growth of various cancercells; and finally the tumorigenicity of the lung cancer cells wasinhibited through the treatment by Ad5CMV-p53 but not a control virusindicating that the novel p53 protein delivery and preparation hasastonishing therapeutic efficacy.

The invention therefore concerns adenovirus vector constructs thatinvolve using Adenovirus to carry tumor suppressor genes such as p53,anti-sense oncogenes and other related genes for human cancer therapy.In one embodiment recombinant Adenovirus virions or particlesincorporating such vectors, and pharmacological formulations thereof,which comprise a recombinant insert including an expression regionencoding wild type p53, by which vectors are capable of expressing p53in human metastatic cells are encompassed. The p53 expression region inthe vector may comprise a genomic sequence, but for simplicity, it iscontemplated that one will generally prefer to employ a p53 cDNAsequence as these are readily available in the art and more easilymanipulated. The recombinant insert of the vector will also generallycomprise a promoter region and a polyadenylation signal, such as an SV40or protamine gene polyadenylation signal.

In preferred embodiments, it is contemplated that one will desire toposition the p53 expression region under the control of a strongconstitutive promoter such as a CMV promoter, viral LTR, RSV, or SV40promoter, or a promoter associated with genes that are expressed at highlevels in mammalian cells such as elongation factor-1 or actinpromoters. Currently, the most preferred promoter is the cytomegalovirus(CMV) IE promoter.

The p53 gene or cDNA may be introduced into recombinant adenovirus inaccordance with the invention simply by inserting or adding the p53coding sequence into a viral genome which lacks E1B. However, thepreferred adenoviruses will be replication defective viruses in which aviral gene essential for replication and/or packaging has been deletedfrom the adenoviral vector construct, allowing the p53 expression regionto be introduced in its place. Any gene in addition to E1B, whetheressential (e.g., E1A, E2 and E4) or non-essential (e.g., E3) forreplication, may be deleted and replaced with p53.

Particularly preferred are those vectors and virions in which the E1Aand E1B regions of the adenovirus vector have been deleted and the p53expression region introduced in their place, as exemplified by thegenome structure of FIG. 1.

Techniques for preparing replication defective adenoviruses are wellknown in the art, as exemplified by Ghosh-Choudhury and Graham (1987);McGrory et al. (1988); and Gluzman et al. (1982), each incorporatedherein by reference. It is also well known that various cell lines maybe used to propagate recombinant adenoviruses, so long as theycomplement any replication defect which may be present. A preferred cellline is the human 293 cell line, but any other cell line that ispermissive for replication, i.e., in the preferred case, which expressesE1A and E1B may be employed. Further, the cells can be propagated eitheron plastic dishes or in suspension culture, in order to obtain virusstocks thereof.

The invention is not limited to E1-lacking virus and E1-expressing cellsalone. Indeed, other complementary combinations of viruses and hostcells may be employed in connection with the present invention, so longas the p53 vector does not have E1B. Virus lacking functional E2 andE2-expressing cells may be used, as may virus lacking functional E4 andE4-expressing cells, and the like. Where a gene which is not essentialfor replication is deleted and replaced, such as, for example, the E3gene, this defect will not need to be specifically complemented by thehost cell.

Other than the requirement that the adenovirus vectors and virions nothave E1B, their nature is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in themethod of the present invention. This is because Adenovirus type 5 is ahuman adenovirus about which there is significant amount of biochemicaland genetic information known, and which has historically been used formost constructions employing adenovirus as a vector.

Further related aspects of the invention concern novel p53 DNA segments,or expression vectors, and recombinant host cells which incorporate anadenoviral p53 vector prepared in accordance herewith. The DNA segmentsof the invention will generally comprise, in the 5′-3′ direction oftranscription, a cytomegalovirus IE promoter, a structural gene for thewild-type human p53, and an SV40 early polyadenylation signal. Therecombinant adenovirus-containing host cell will generally be aeukaryotic or mammalian host cell, such as a 293 cell, or may be a cellwith a defect in a p53 gene which has been infected with the adenovirusof the invention.

Other embodiments concern pharmaceutical compositions comprising arecombinant adenovirus which encodes wild type p53, dispersed in apharmacologically acceptable solution or buffer. Preferredpharmacologically acceptable solutions include neutral saline solutionsbuffered with phosphate, lactate, Tris, and the like. Of course, onewill desire to purify the adenovirus sufficiently to render itessentially free of undesirable contaminants, such as defectiveinterfering adenovirus particles or endotoxins and other pyrogens suchthat it will not cause any untoward reactions in the animal orindividual receiving the vector construct. A preferred means ofpurifying the vector involves the use of buoyant density gradients, suchas cesium chloride gradient centrifugation.

In still further embodiments, the invention relates to a method forproviding p53 functions, or restoring wild-type p53 protein functions,to a cell deficient in wild-type p53. To achieve this, one would contactthe cell bearing the p53 mutation with an amount of recombinantadenovirus of the invention effective to express wild-type p53 in thecell. This may be achieved by administering a physiologically effectiveamount of a pharmaceutical composition comprising the adenovirus to ananimal or human subject which harbors cells with defective p53, such as,e.g., cancer cells. Therefore, the present invention also encompasseseffective methods for treating human malignancies such as breast andlung cancer.

In another embodiment of the invention the p53 expressing adenovirus isused to prevent malignant and even metastatic growth. In one embodimentthe recombinant p53 expressing adenovirus is used to inhibit theuncontrolled growth of cells that have mutations of the p53 gene. In amore preferred embodiment the p53 expressing adenovirus inhibits thetumorigenicity and growth of H358 cells, but any other cell that is anindicator of p53 function may be used.

In a further embodiment the p53 expressing virus is used to preventorthotopic lung tumor growth when the virus is instilledintratracheally. The Ad5CMV-p53 virus yielded encouraging results in thenude mouse tests. The virus inhibited tumorigenicity of virus-infectedH358 cells, a cell that normally produces a significant tumor mass. Thevirus also prevented orthotopic lung cancer growth when the virus wasinstilled intratracheally following the intratracheal inoculation ofH226Br cells confirming the in vitro effects of Ad5CMV-p53 on the lungcancer cells. The tumorigenicity of the lung cancer cells was inhibitedthrough the treatment by Ad5CMV-p53 but not by the control virusAd5/RSV/GL2, indicating that the p53 protein has therapeutic efficacy.It will be understood by those skilled in the art that other methods ofviral delivery are encompassed by the invention.

While aspects of the invention are exemplified through the use of p53constructs in connection with restoring normal cell function and for usein cancer treatment, it is proposed that the invention is generallyapplicable to any situation where one desires to achieve high levelexpression of a tumor suppressor protein in a target or host cellthrough the use of recombinant adenovirus. For example, in the contextof cancer treatment modalities, a particular example in addition to p53replacement that is contemplated by the inventors is the introduction ofthe retinoblastoma gene (rb), anti-sense oncogenes (c-myc or c-ras), andother related genes for human cancer therapy.

It should be pointed out that because the adenovirus vector employed isreplication defective, it will not be capable of replicating in thecells, such as cancer cells, that are ultimately infected. Thus, wherecontinued treatment in certain individuals is required, such as at thebeginning of therapy, it may be necessary to reintroduce the virus aftera certain period, for example, 6 months or a year.

The adenoviral vectors of the present invention will also have utilityin embodiments other than those connected directly with gene therapy.Alternative uses include, for example, in vitro analyses and mutagenesisstudies of various p53 genes, and the recombinant production of proteinsfor use, for example, in antibody generation or other embodiments. Inembodiments other than those connected with human therapy, including allthose concerned with further defining the molecular activity of p53,other related viruses may even be employed to deliver p53 to a cell.Those belonging to the herpes family, e.g., herpes simplex virus (HSV),Epstein-Barr Virus (EBV), cytomegalovirus (CMV) and pseudorabies virus(PRV) would be suitable.

A different aspect of the present invention concerns simplifiedprocedures for producing any type of recombinant adenovirus which avoidusing the inefficient calcium phosphate transfection and tedious plaqueassays. To produce recombinant adenovirus in accordance with the presentinvention, one would generally introduce an adenovirus plasmid and anexpression vector into a suitable host cell by liposome-mediatedtransfection, and then analyze the cultured host cell for the presenceof a cytopathic effect (CPE), which is indicative of homologousrecombination and virus production. It is the increase in transfectionefficiency of the first step which renders the second and advantageousCPE step possible.

A preferred composition for use in the liposome-mediated transfection isDOTAP (N-[1-(2,3-dideoyloxy)propyl]-N,N,N-trimethyl-amrnmoniummethysulfate) which is commerciallyavailable. CPE is a directly observable phenomenon, which may beassessed using phase contrast microscopy. CPE describes the morphologicfeatures of Adenovirus cytotoxicity that begin with the shrinking of thelytically infected cell and conclude with the formation of a lyticplaque. A particular advantage of this method is that viral propagationis readily determined after a 10 to 14 day incubation. This is asignificant improvement over the calcium phosphate-mediated transfectionand subsequent plaque assays require at least 14 and usually up to aminimum of 21 days, and frequently up to several weeks before theresults can be assessed.

In certain embodiments, the method of the invention may be used inconnection with adenovirus plasmids which are replication-defective,along with a host cell which complements the defect, as exemplified byE1-lacking plasmids and 293 cells. Adenovirus plasmids which lackfunctional E1A and E1B and which incorporate a p53 expression region areused herein in working examples of the invention, but any expressionregion may be incorporated into a recombinant adenovirus in this manner.The precise methodological aspects may be varied as desired; however, itis contemplated that the use of MEM media will be preferred in certaincases.

These new methods may be combined with PCR analysis to confirm thepresence of the correctly recombined virus. PCR is well known to thosein the art, as disclosed in U.S. Pat. No. 4,683,195, incorporated hereinby reference. To use PCR in connection with the invention, one wouldobtain DNA from the supernatant of cells exhibiting a cytopathic effectand analyze the DNA by PCR using two pairs of primers, one expressionvector-specific and the other adenoviral genome-specific DNA primers.Vector- or insert-specific DNA is, by definition, a gene segment whichis part of the DNA encoding the polypeptide or RNA one ultimatelydesires to be expressed, as illustrated by p53 DNA expression as mRNAand protein production. Adenovirus genome specific DNA may be any partof the genome that is expressed during the stage of propagation beingmonitored.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Scheme for generation of recombinant p53 adenovirus. The p53expression cassette was inserted between the Xba I and Cla I sites ofpXCJL.1. The p53 expression vector (pEC53) and the recombinant plasmidpJM17 were cotransfected into 293 cells. The transfected cells weremaintained in medium until the onset of the cytopathic effect.Identification of newly generated p53 recombinant adenoviruses(Ad5CMV-p53) by PCR analysis of the DNA using DNA templates preparedfrom the CPE supernatants treated with Proteinase K and phenolextraction.

FIG. 2A shows a map used for the structural analysis of Ad5CMV-p53 DNA.A map of Ad5CMV-p53 genomic DNA, with the locations of the p53expression cassette, the PCR primers, and the restriction sites. Thegenome size is about 35.4 kb, divided into 100 maps units (1 m.u.=0.35kb). The p53 expression cassette replaced the E1 region (1.3-9.2 m.u.)of the Ad5 genome. Primer 1 is located in the first intron downstream ofthe human CMV major IE gene promoter. Primer 2 is located in SV40 earlypolyadenylation signal. Both of the primers, 15-20 bp away from the p53cDNA insert at both ends, define a 1.40 kb PCR product. Primers 3 and 4are located at 11 m.u. and 13.4 m.u. of Ad5 genome, respectively, whichdefine a 0.86 kb viral-genome specific PCR product.

FIG. 2B shows agarose gel analysis of PCR products. Two pairs of primersthat define 1.4-kb (p53) and 0.86-kb (Ad5) DNA fragments were used ineach reaction. DNA templates used in each reaction were pEC53 plasmid(lane 1), Ad5/RSV/GL2 DNA (lane 2), no DNA (lane 3), and Ad5CMV-p53 DNA(lane 4). The lane labeled (M) corresponds to molecular weight markers.

FIG. 2C shows restriction mapping of Ad5CMV-p53 DNA. CsCl-gradientpurified Ad5CMV-p53 DNA samples were digested with no enzyme (U), HindIII (H), Bam HI (B), Eco RI (E), and Cla I (C), respectively, andanalyzed on 1% agarose gel. The lanes labeled (M) are molecular weightmarkers.

FIGS. 3A, 3B, 3C and 3D, observation of cytopathic effects on 293 byrecombinant adenovirus. FIGS. 3A, 3B, 3C and 3D are a series of phasecontrast images (×400) of 293 cells. FIGS. 3A, 3B, 3C and 3D are fourpanels of a single page figure. FIG. 2A, before transfection; FIG. 3B,negative control on day 12 posttransfection; FIG. 3C, onset of CPE onday 12 posttransfection; FIG. 3D, completion of CPE on day 14post-transfection.

FIGS. 4A, 4B, 4C, and 4D, immunohistology of cells infected withrecombinant adenoviruses. FIGS. 4A, 4B, 4C and 4D are a series ofimmunohistological images of H358 cells. FIGS. 4A, 4B, 4C and 4D arefour panels of a single page figure. Infectivity of Ad5CMV-p53 in H358cells. H358 cells were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 50PFU/cell for 24 h. Medium alone was used as a mock infection. Theinfected cells were analyzed by immunostainings. FIG. 4A is a mockinfection probed with anti-p53 antibody. FIG. 4B are cells infected withthe Ad5/RSV/GL2 control and probed with anti-p53 antibody. FIG. 4C areAd5CMV-p53 infected cells probed with an unrelated antibody (MOPC-21).FIG. 4D are cells Ad5CMV-p53 infection probed with anti-p53 antibody.The anti-p53 antibody used was Pab 1801, and the avidin-biotin methodwas used for staining.

FIG. 5A shows a Coomassie-blue stained SDS-PAGE gel comparing therelative level of expression of exogenous p53 in H358 cells. H358 cellsamples that were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 30 PFU/cellwere prepared 24 and 72 h after infection. Coomassie blue staining of anSDS-PAGE analysis, showing relative quantities of protein samplesloaded. Lanes 1 and 4 contain the samples of the Ad5/RSV/GL2-infectedcells. Lanes 2 and 3 contain the samples of the cells infected with twoindividual stocks of Ad5CMV-p53 at 24 h after infection. Lanes 5 and 6are the Ad5CMV-p53-infected cell samples collected at 72 h afterinfection. Lane 7 is mock-infected H358 sample 72 h after infection.Lane M, prestained molecular weight markers in kDa (GIBCO-BRL).

FIG. 5B shows a Western blot analysis of the identical lane setting gelas that of the SDS-PAGE in FIG. 5A. The relative levels of expression ofp53 were analyzed by Western blotting using anti-p53. Primary antibodiesused were monoclonal antibodies against p53 protein (PAb 1801, OncogeneScience Inc.) and β-actin (Amersham Inc.). The HRP-conjugated secondantibody and ECL developer were from Amershem Inc. viral-infected H358cells by Western Blotting. Western blot of FIG. 5B have an equivalentsetup and order to those in FIG. 5A.

FIG. 6 is a time course of the p53 expression, determined by Westernblotting. Multiple dishes of H358 cells were infected with Ad5CMV-p53 at10 PFU/cell. Cell lysates were prepared at indicated time points afterinfection. Western blotting was probed with anti-p53 and anti-actinantibodies simultaneously. The lanes designated ‘C’ represent negativecontrols. The histogram represents the relative quantities of p53 asdetermined by a densitometer.

FIG. 7A shows the growth curve of virally-infected human lung cancercells of cell lines H358. Cells were inoculated at 105 cells per dish(60 mm) and 6 dishes per cell line. After 24 hours, the cells wereinfected with Ad5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (Multiplicity ofinfection, i.e., PFU/cell). After infection cells were counted daily for6 days. The growth curves represent data obtained from 4 separatestudies.

FIG. 7B shows the growth curve of virally-infected human lung cancercells of cell line H322. Cells were inoculated at 10⁵ cells per dish (60mm) and 6 dishes per cell line. After 24 hours, the cells were infectedwith Ad5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (Multiplicity of infection,i.e., PFU/cell). After infection cells were counted daily for 6 days.The growth curves represent data obtained from 4 separate studies.

FIG. 7C shows the growth curve of virally-infected human lung cancercells of cell line H460. Cells were inoculated at 10⁵ cells per dish (60mm) and 6 dishes per cell line. After 24 hours, the cells were infectedwith Ad5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (Multiplicity of infection,i.e., PFU/cell). After infection cells were counted daily for 6 days.The growth curves represent data obtained from 4 separate studies.

FIG. 8 shows a flow chart of tests of Ad5CMV-p53 in orthotopic lungcancer model. The dosages and schedule of treatment of nude miceinnoculated with H226Br cells and viruses are summarized in the flowchart.

FIGS. 9A, 9B, 9C, and 9D are samples of the lung and mediastinumdissection from treated and control mice. FIGS. 9A, 9B, 9C, and 9D arefour panels of a single figure. The mice were sacrificed at the end ofthe 6-week posttreatment period. The lung and mediastinum tissues weredissected for evaluation of tumor formation. FIG. 9A is a sample ofmediastinal block from a normal nude mice; FIG. 9B is the mediastinalblock sample from the vehicle (PBS)-treated mice; FIG. 9C is themediastinal block sample from the Ad5CMV-p53-treated mice; FIG. 9D isthe mediastinal block sample from the Ad5/RSV/GL2-treated mice. Arrowsindicate the tumor masses.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Molecular Events in Lung Cancer Development

Studies carried out by the present inventors has identified criticalmolecular events leading to the development and progression of cancer.This enabled the inventors to develop new methods for restoring certainnormal protein functions so that the malignant phenotype can besuppressed in vivo.

The most common lung cancer histologies (80%) are grouped under the termnon-small-cell lung cancer (NSCLC) and include squamous, adenocarcinoma,and large-cell undifferentiated. Many of the current data on themolecular biology of lung cancer come from the study of the moreuncommon small-cell lung cancer (SCLC). SCLC can be distinguished fromNSCLC by the neuroendocrine features of the cells; SCLC is veryresponsive to chemotherapy but recurs rapidly after treatment. NSCLCalso may serve as a model for other carcinogen-induced epithelialcancers. The approaches and observations developed in this study may beapplicable to other types of epithelial cancers.

Abundant evidence has accumulated that the process of malignanttransformation is mediated by a genetic paradigm. The major lesionsdetected in cancer cells occur in dominant oncogenes and tumorsuppressor genes. Dominant oncogenes have alterations in a class ofgenes called proto-oncogenes, which participate in critical normal cellfunctions, including signal transduction and transcription. Primarymodifications in the dominant oncogenes that confer the ability totransform include point mutations, translocations, rearrangements, andamplification. Tumor suppressor genes appear to require homozygous lossof function, by mutation, deletion, or a combination of these fortransformation to occur. Some tumor suppressor genes appear to play arole in the governance of proliferation by regulation of transcription.Modification of the expression of dominant and tumor suppressoroncogenes is likely to influence certain characteristics of cells thatcontribute to the malignant phenotype.

Despite increasing knowledge of the mechanisms involved inoncogene-mediated transformation, little progress has occurred indeveloping therapeutic strategies that specifically target oncogenes andtheir products. Initially, research in this area was focused on dominantoncogenes, as these were the first to be characterized. DNA-mediatedgene transfer studies showed acquisition of the malignant phenotype bynormal cells following the transfer of DNA from malignant human tumors.

B. p53 and p53 Mutations in Cancer

P53 is currently recognized as a tumor suppressor gene (Montenarh,1992). High levels have been found in many cells transformed by chemicalcarcinogenesis, ultraviolet radiation, and several viruses, includingSV40. The p53 gene is a frequent target of mutational inactivation in awide variety of human tumors and is already documented to be the mostfrequently-mutated gene in common human cancers (Mercer, 1992). It ismutated in over 50% of human NSCLC (Hollestein et al., 1991) and in awide spectrum of other tumors.

The p53 gene encodes a 375-amino-acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue.Interestingly, wild-type p53 appears to be important in regulating cellgrowth and division. Overexpression of wild-type p53 has been shown insome cases to be anti-proliferative in human tumor cell lines. Thus p53can act as a negative regulator of cell growth (Weinberg, 1991) and maydirectly suppress uncontrolled cell growth or indirectly activate genesthat suppress this growth. Thus, absence or inactivation of wild typep53 may contribute to transformation. However, some studies indicatethat the presence of mutant p53 may be necessary for full expression ofthe transforming potential of the gene.

Although wild-type p53 is recognized as a centrally important growthregulator in many cell types, its genetic and biochemical traits appearto have a role as well. Mis-sense mutations are common for the p53 geneand are essential for the transforming ability of the oncogene. A singlegenetic change prompted by point mutations can create carcinogenic p53.Unlike other oncogenes, however, p53 point mutations are known to occurin at least 30 distinct codons, often creating dominant alleles thatproduce shifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). The p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 does not affect the growth of cellswith endogenous p53. Thus, such constructs might be taken up by normalcells without adverse effects.

It is thus possible that the treatment of p53-associated cancers withwild type p53 may reduce the number of malignant cells. However, studiessuch as those described above are far from achieving such a goal, notleast because DNA transfection cannot be employed to introduce DNA intocancer cells within a patients' body.

C. Gene Therapy Techniques

There have been several experimental approaches to gene therapy proposedto date, but each suffer from their particular drawbacks (Mulligan,1993). As mentioned above, basic transfection methods exist in which DNAcontaining the gene of interest is introduced into cellsnon-biologically, for example, by permeabilizing the cell membranephysically or chemically. Naturally, this approach is limited to cellsthat can be temporarily removed from the body and can tolerate thecytotoxicity of the treatment, i.e. lymphocytes. Liposomes or proteinconjugates formed with certain lipids and amphophilic peptides can beused for transfection, but the efficiency of gene integration is stillvery low, on the order of one integration event per 1,000 to 100,000cells, and expression of transfected genes is often limited to days inproliferating cells or weeks in non proliferating cells. DNAtransfection is clearly, therefore, not a suitable method for cancertreatment.

A second approach capitalizes on the natural ability of viruses to entercells, bringing their own genetic material with them. Retroviruses havepromise as gene delivery vectors due to their ability to integrate theirgenes into the host genome, transferring a large amount of foreigngenetic material, infecting a broad spectrum of species and cell typesand of being packaged in special cell-lines. However, three majorproblems hamper the practical use of retrovirus vectors. First,retroviral infectivity depends on the availability of the viralreceptors on the target surface. Second, retroviruses only integrateefficiently into replicating cells. And finally, retroviruses aredifficult to concentrate and purify.

D. Adenovirus Constructs for use in Gene Therapy

Human adenoviruses are double-stranded DNA tumor viruses with genomesizes of approximate 36 kb (Tooza, 1981). As a model system foreukaryotic gene expression, adenoviruses have been widely studied andwell characterized, which makes them an attractive system fordevelopment of adenovirus as a gene transfer system. This group ofviruses is easy to grow and manipulate, and they exhibit a broad hostrange in vitro and in vivo. In lytically infected cells, adenovirusesare capable of shutting off host protein synthesis, directing cellularmachineries to synthesize large quantities of viral proteins, andproducing copious amounts of virus.

The E1 region of the genome includes E1A and E1B which encode proteinsresponsible for transcription regulation of the viral genome, as well asa few cellular genes. E2 expression, including E2A and E2B, allowssynthesis of viral replicative functions, e.g. DNA-binding protein, DNApolymerase, and a terminal protein that primes replication. E3 geneproducts prevent cytolysis by cytotoxic T cells and tumor necrosisfactor and appear to be important for viral propagation. Functionsassociated with the E4 proteins include DNA replication, late geneexpression, and host cell shutoff. The late gene products include mostof the virion capsid proteins, and these are expressed only after mostof the processing of a single primary transcript from the major latepromoter has occurred. The major late promoter (MLP) exhibits highefficiency during the late phase of the infection (Stratford-Perricaudetand Perricaudet, 1991a).

As only a small portion of the viral genome appears to be required incis (Tooza, 1981), adenovirus-derived vectors offer excellent potentialfor the substitution of large DNA fragments when used in connection withcell lines such as 293 cells. Ad5-transformed human embryonic kidneycell line (Graham, et al., 1977) have been developed to provide theessential viral proteins in trans. The inventors thus reasoned that thecharacteristics of adenoviruses rendered them good candidates for use intargeting cancer cells in vivo (Grunhaus & Horwitz, 1992).

Particular advantages of an adenovirus system for delivering foreignproteins to a cell include (i) the ability to substitute relativelylarge pieces of viral DNA by foreign DNA; (ii) the structural stabilityof recombinant adenoviruses; (iii) the safety of adenoviraladministration to humans; and (iv) lack of any known association ofadenoviral infection with cancer or malignancies; (v) the ability toobtain high titers of the recombinant virus; and (vi) the highinfectivity of Adenovirus.

Further advantages of adenovirus vectors over retroviruses include thehigher levels of gene expression. Additionally, adenovirus replicationis independent of host gene replication, unlike retroviral sequences.Because adenovirus transforming genes in the E1 region can be readilydeleted and still provide efficient expression vectors, oncogenic riskfrom adenovirus vectors is thought to be negligible (Grunhaus & Horwitz,1992).

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus which is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Relatively large foreign proteins can beexpressed when additional deletions are made in the adenovirus genome.For example, adenoviruses deleted in both E1 and E3 regions are capableof carrying up to 10 Kb of foreign DNA and can be grown to high titersin 293 cells (Stratford-Perricaudet and Perricaudet, 1991a).Surprisingly persistent expression of transgenes following adenoviralinfection has also been reported.

Adenovirus-mediated gene transfer has recently been investigated as ameans of mediating gene transfer into eukaryotic cells and into wholeanimals. For example, in treating mice with the rare recessive geneticdisorder ornithine transcarbamylase (OTC) deficiency, it was found thatadenoviral constructs could be employed to supply the normal OTC enzyme.Unfortunately, the expression of normal levels of OTC was only achievedin 4 out of 17 instances (Stratford-Perricaudet et al., 1991b).Therefore, the defect was only partially corrected in most of the miceand led to no physiological or phenotypic change. These type of resultstherefore offer little encouragement for the use of adenoviral vectorsin cancer therapy.

Attempts to use adenovirus to transfer the gene for cystic fibrosistransmembrane conductance regulator (CFTR) into the pulmonary epitheliumof cotton rats have also been partially successful, although it has notbeen possible to assess the biological activity of the transferred genein the epithelium of the animals (Rosenfeld et al., 1992). Again, thesestudies demonstrated gene transfer and expression of the CFTR protein inlung airway cells but showed no physiologic effect. In the 1991 Sciencearticle, Rosenfeld et al. showed lung expression of α1-antitrypsinprotein but again showed no physiologic effect. In fact, they estimatedthat the levels of expression that they observed were only about 2% ofthe level required for protection of the lung in humans, i.e., far belowthat necessary for a physiologic effect.

The gene for human α₁-antitrypsin has been introduced into the liver ofnormal rats by intraportal injection, where it was expressed andresulted in the secretion of the introduced human protein into theplasma of these rats (Jaffe et al., 1992). However, and disappointingly,the levels that were obtained were not high enough to be of therapeuticvalue.

These type of results do not demonstrate that adenovirus is able todirect the expression of sufficient protein in recombinant cells toachieve a physiologically relevant effect, and they do not, therefore,suggest a usefulness of the adenovirus system for use in connection withcancer therapy. Furthermore, prior to the present invention, it wasthought that p53 could not be incorporated into a packaging cell, suchas those used to prepare adenovirus, as it would be toxic. As E1B ofadenovirus binds to p53, this was thought to be a further reason whyadenovirus and p53 technology could not be combined.

E. p53-Adenovirus Constructs and Tumor Suppression

The present invention provides cancer gene therapy with a new and moreeffective tumor suppressor vector. This recombinant virus exploits theadvantages of adenoviral vectors, such as high titer, broad targetrange, efficient transduction, and non-integration in target cells. Inone embodiment of the invention, a replication-defective,helper-independent adenovirus is created that expresses wild type p53(Ad5CMV-p53) under the control of the human cytomegalovirus promoter.

Control functions on expression vectors are often provided from viruseswhen expression is desired in mammalian cells. For example, commonlyused promoters are derived from polyoma, adenovirus 2 and simian virus40 (SV40). The early and late promoters of SV40 virus are particularlyuseful because both are obtained easily from the virus as a fragmentwhich also contains the SV40 viral origin of replication. Smaller orlarger SV40 fragments may also be used provided there is included theapproximately 250 bp sequence extending from the HindIII site toward theBglI site located in the viral origin of replication. Further, it isalso possible, and often desirable, to utilize promoter or controlsequences normally associated with the included gene sequence, providedsuch control sequences are compatible with the host cell systems.

An origin of replication may be provided by construction of the vectorto include an exogenous origin, such as may be derived from SV40 orother viral (e.g., polyoma, adeno, VSV, BPV) source, or may be providedby the host cell chromosomal replication mechanism. If the vector isintegrated into the host cell chromosome, the latter is oftensufficient.

The design and propagation of the preferred p53 adenovirus is diagramedin FIG. 1. In connection with this, an improved protocol has beendeveloped for propagating and identifying recombinant adenovirus(discussed below). After identification, the p53 recombinant adenoviruswas structurally confirmed by the PCR analysis, as indicated in FIG. 2.After isolation and confirmation of its structure, the p53 adenoviruswas used to infect human lung cancer cell line H358, which has ahomozygous p53 gene deletion. Western blots showed that the exogenousp53 protein was expressed at a high level (FIG. 4 and FIG. 5) and peakedat day 3 after infection (FIG. 6).

It was also shown in a p53 point mutation cell line H322 that the mutantp53 was down regulated by the expression of the exogenous p53. As anexperimental control, a virion (Ad5/RSV/GL2) that had a structuralsimilarity to that of Ad5CMV-p53 was used. This virion contained aluciferase CDNA driven by Rous sarcoma virus LTR promoter in theexpression cassette of the virion. Neither p53 expression nor change inactin expression was detected in cells infected by the virionAd5/RSV/GL2. Growth of the H358 cells infected with Ad5CMV-p53 wasgreatly inhibited in contrast to that of noninfected cells or the cellsinfected with the control virion (FIG. 7A). Growth of H322 cells wasalso greatly inhibited by the p53 virion (FIG. 7B), while that of humanlung cancer H460 cells containing wild-type p53 was less affected (FIG.7C).

Ad5CMV-p53 mediated a strong inhibitory effect on lung cancer cellgrowth in vitro. Growth inhibition was not as evident when the cellswere infected with Ad5CMV-p53 at MOI lower than 1 PFU/cell, whereas, atMOI higher than 100 PFU/cell, cytotoxicity could be observed even withcontrol virus Ad5/RSV/GL2. In our studies, the optimal dose for growthrate studies was 10-50 PFU/cell. Within this dose range, cell growthinhibition was attributable to the expressed p53 protein.

Tests in nude mice demonstrated that tumorigenicity of theAd5CMV-p53-treated H358 cells was greatly inhibited. In a mouse model oforthotopic human lung cancer, the tumorigenic H226Br cells, with a pointmutation in p53, were inoculated intratracheally 3 days prior to thevirus treatment. Intratracheal instillation of Ad5CMV-p53 preventedtumor formation in this model system suggesting that the modifiedadenovirus is an efficient vector for mediating transfer and expressionof tumor suppressor genes in human cancer cells and that the Ad5CMV-p53virus may be further developed into a therapeutic agent for use incancer gene therapy.

Ad5CMV-p53 mediated a high level of expression of the p53 gene in humanlung cancer cells as demonstrated by Western blot analysis. Exogenousp53 protein was approximately 14 times more abundant than the endogenouswild-type p53 in H460 cells and about two to four times more abundantthan the β-actin internal control in H358 cells. The high level ofexpression may be attributed to (1) highly efficient gene transfer, (2)strong CMV promoter driving the p53 CDNA, and (3) adenoviral E1 enhancerenhancing the p53 CDNA transcription. The duration of p53 expressionafter infection was more than 15 days in H358 cells. However, there wasa rapid decrease in expression after postinfection day 5. PCR analysisof the DNA samples from the infected H358 cells showed a decrease of theviral DNA level with the decreased protein level, indicating the loss ofviral DNA during the continuous growth of cancer cells in vitro.

The decrease in p53 expression may also have resulted from cellularattenuation of the CMV promoter that controls p53 expression, since thephenomenon of host cell-mediated CMV promoter shut off has been reportedpreviously (Dai, et al., 1992). Adenoviral vectors are nonintegrativegene transfer vectors and therefore the duration of gene expressiondepends upon a number of factors, including the host cells, the genestransferred, and the relevant promoter. Crystal and co-workers showedlow level expression of the cystic fibrosis transmembrane conductanceregulator gene in cotton rat epithelial cells was detectable 6 weeksafter infection (Rosenfeld, et al., 1992). Perricaudet's laboratorydemonstrated minimal expression of minidystrophin gene in mdx mousemuscle lasted for more than 3 months after infection. The short-termhigh level expression of the wild-type p53 protein observed in thepresent study may have the beneficial effect of reducing possible sideeffects on normal cells following in vivo treatment with Ad5CMV-p53.

The studies disclosed herein indicate that the p53 recombinantadenovirus possesses properties of tumor suppression, which appear tooperate by restoring p53 protein function in tumor cells. These resultsprovide support for the use of the Ad5CMV-p53 virion as a therapeuticagent for cancer treatment.

F. Improved Protocol for Propagating and Identifying RecombinantAdenovirus

Recombinant adenovirus as a new gene delivery system has many potentialapplications in gene therapy and vaccine development. Propagation ofrecombinant adenovirus is therefore an important molecular biologicaltool. The existing methods for propagating recombinant adenovirus usecalcium phosphate precipitation-mediated transfection into 293 cells andsubsequent plaque assays on the transfected cells. The transfectionefficiency associated with this method needs to be improved and, also,the procedure could be simplified.

Prior to the present invention, propagation of recombinant adenoviruswas conventionally carried out by calcium phosphate-mediatedtransfection. This procedure involves exposing cells to vector orplasmid DNA in calcium phosphate for several hours prior to a briefshock treatment, e.g., one minute in 15% glycerol. This methods suffersfrom the significant drawback of resulting in only low levels of DNAbeing incorporated into the cell, i.e., it is a very inefficient meansof transfection. Viral propagation was also normally indicated by theappearance of plaques which are observed as clear, round areas aroundlysed cells indicating cell lysis caused by virus propagation.

The inventors have developed a novel procedure for producing adenoviruswhich significantly improves the transfection efficiency and alsosimplifies selection. The inventors have discovered that a combinationof liposome-mediated transfection, such as DOTAP-mediated transfection,with the observation of cytopathic effect (CPE) leads to both improvedefficiency and rapid and simplified detection. In the new procedure,liposome DOTAP-mediated gene transfer is used to transduce an expressionvector and recombination plasmid into 293 cells. The transfected cells,instead of being overlaid with 0.5% agarose for plaque assays, are thenmaintained continually in MEM medium for observation of cytopathiceffect (CPE).

In two studies using the new method, 2 wells out of a 24-well plate and3 dishes out of five 60-mm dishes generated CPE at days 10 and 12 aftercotransfection, respectively. In contrast, using the calcium-phosphateprecipitation method, no recombinant virus was obtained in three trialsfrom the cotransfection with twenty 60-mm dishes in each experiment.Using CAT assays in Hep G2 and HeLa cell lines, DOTAP-mediatedtransfection resulted in 5 fold higher CAT activity than calciumphosphate transfection.

Elimination of the agarose overlay after cotransfection also simplifiedthe procedure. The endpoint of the study, propagation of virus, becomesmuch more simple and clear by directly observing the CPE instead ofidentifying plaques, which is usually unclear and difficult to determineafter 10-14 day incubation. FIG. 3 shows the cell culture with CPE incomparison with the cell culture without CPE.

The inventors have also developed a rapid technique to determineadenovirus titers using PCR. The direct PCR analysis of DNA samples fromthe supernatant of the cell cultures with CPE conveniently uses twopairs of primers, one to amplify insert-specific and the other toamplify viral genome-specific sequences. The inventors have shown thatadenoviruses released into the cell culture medium are detectable byPCR, thereby allowing one to use as little as about 50 μL of supernatantto prepare DNA templates.

Identification of the insert-specific and viral genome-specific DNAsequences resulting from PCR amplification may be determined by, forexample, analysis of PCR amplified products on agarose gels. Bandscorresponding to insert-specific DNA and viral genome-specific DNA, andalso the primers, may be identified by comparison with the appropriatestandard markers.

Where PCR is employed to amplify insert-specific and viralgenome-specific gene products, one will first prepare a primer specificfor the sequences to be amplified. Efficient and selective amplificationis achieved by employing two primer pairs: one to amplify a definedsection of the insert-specific product, and the other to define asegment of the viral genome-specific product. By way of example only, acassette for expression of p53 has been constructed with a humancytomegalovirus (CMV) promoter and SV40 early polyadenylation signal.The first primer set will include a primer located in the first introndownstream of the human CMV major IE gene promoter while the otherprimer of the first primer set will be located in SV40 earlypolyadenylation signal. Ideally, both of these primers are 15 to 20 basepairs away from the cDNA insert which, in the illustrative example, isp53.

A defined PCR product should be selected, for example a 1.40 kb p53cDNA. As an example, the second set of primers may be located at 11 M.U.and 13.4 M.U. of the Ad5 genome to define a 0.86 kb viral genomespecific PCR product.

Primer selection is well known to those of skill in the art. One mayconstruct primers for amplification of selected portions to DNAsequences whose base pair sequence is known. Primers hybridize to DNAand serve as initiation sites for synthesis of a portion of the gene.Nucleotide primers are designed to bind at separate sites on opposingduplex strains thereby defining the intervening sequence as the portionto be amplified. Nucleic acid molecules to be employed as primers willgenerally include at least a 10 base pair sequence which will becomplementary to the DNA segment one desires to amplify. The 10 basepair size is selected as a general lower limit in that sizes smallerthan 10 bases may not effectively hybridize and stabilization may becomea problem. Preferably, sizes of 15-20 are utilized and in a preferredaspect of the invention the primer pairs shown in FIG. 1 are employedwhere size is 19 or 20 base pairs.

G. Patients and Treatment Protocols

The inventors propose that the regional delivery of adenoviral-p53 geneconstructs to lung cancer cells in patients with p53-linked cancers,such as unresectable obstructing endobronchial cancers, will be a veryefficient method for delivering a therapeutically effective gene tocounteract the clinical disease. It is proposed that this approach is asignificant improvement on current cancer therapies which rely onattempts to kill or remove the last cancer cell. As tumor cell dormancyis an established phenomenon, this makes effective killing highlyunlikely.

It is anticipated that the uptake of the adenovirus constructs by NSCLCcells will decrease the rate of proliferation of these cells. This wouldincrease the length of time the affected lung would remain expanded,prevent regrowth of the endobronchial tumor, and prolong the patient'ssurvival.

Patients with unresectable endobronchial tumor recurrence that ispartially or completely obstructing the airway and that have failed orare unable to receive external beam radiotherapy will be considered forthis protocol. Existing therapies for this condition offer onlyshort-term palliation. Most patients have recurred despite external beamradiotherapy. It may be possible to insert a brachytherapy catheter andadminister additional radiotherapy. Patients receiving this treatmenthave a median survival of 6 months. Patients failing brachytherapy wouldalso be eligible to receive gene therapy. Tumor can be removed from theairway with the laser or biopsy forceps. This can be done in conjunctionwith injection of the adenoviral constructs thus decreasing the volumethat must be injected. The administration of the viral constructs wouldnot preclude the patient from receiving other palliative therapy if thetumor progresses.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Construction of p53 Expression Vector

This example describes the construction of a p53 expression vector. Thisvector is constructed as indicated and is used to replace the E1 region(1.3-9.2 m.u.) of the Adenovirus strain Ad5 genome and employed toconstruct the Adenovirus virion described in Example 2.

The p53 expression cassette shown in FIG. 1, which contains humancytomegalovirus (CMV) promoter (Boshart, et al., 1985), p53 cDNA, andSV40 early polyadenylation signal, was inserted between the Xba I andCla I sites of pXCJL1 (provided by Dr. Frank L. Graham, McMasterUniversity, Canada).

The genome size is about 35.4 kb, divided into 100 map units (1m.u.=0.35 kb) The p53 expression cassette replaced the E1 region(1.3-9.2 m.u.) of the Ad5 genome.

Primer 1 has the sequence 5′-GGCCCACCCCCTTGGCTTC-3′ (SEQ ID NO:1) and islocated in the first intron downstream of the human CMV major IE genepromoter (Boshart, et al., 1985) Primer 2 has the sequence5′-TTGTAACCATTATAAGCTGC-3′ (SEQ ID NO:2) and is located in SV40 earlypolyadenylation signal. Both of the primers, 15-20 bp away from the p53cDNA insert at both ends, define a 1.40 kb PCR product. Primer 3 has thesequence 5′-TCGTTTCTCAGCAGCTGTTG-3′ (SEQ ID NO:3) and primer 4 has thesequence 5′-CATCTGAACTCAAAGCGTGG-3′(SEQ ID NO:4) and are located at 11m.u. and 13.4 m u. of the Ad5 genome, respectively, which define a 0.86kb viral-genome specific PCR product.

EXAMPLE 2 Generation and Propagation of Recombinant p53 Adenovirus

This example describes one method suitable for generatinghelper-independent recombinant adenoviruses expressing p53. Themolecular strategy employed to produce recombinant adenovirus is basedupon the fact that, due to the packaging limit of adenovirus, pJM17cannot form virus on its own. Therefore, homologous recombinationbetween the p53 expression vector plasmid and pJM17 within a transfectedcell results in a viable virus that can be packaged only in cells whichexpress the necessary adenoviral proteins.

The method of this example utilizes 293 cells as host cells to propagateviruses that contain substitutions of heterologous DNA expressioncassettes at the E1 or E3 regions. This process requires cotransfectionof DNA into 293 cells. The transfection largely determines efficiency ofviral propagation. The method used for transfection of DNA into 293cells prior to the present invention was usually calcium-phosphate/DNAcoprecipitation (Graham and van der Eb, 1973). However, this methodtogether with the plaque assay is relatively difficult and typicallyresults in low efficiency of viral propagation. As illustrated in thisexample, transfection and subsequent identification of infected cellswere significantly improved by using liposome-mediated transfection,when identifying the transfected cells by cytopathic effect (CPE).

The 293 cell line was maintained in Dulbecco's modified minimalessential medium supplemented with 10% heat-inactivated horse serum. Thep53 expression vector and the plasmid pJM17 (McGrory, et al., 1988) forhomologous recombination were cotransfected into 293 cells byDOTAP-mediated transfection according to the manufacture's protocol(Boehringer Mannheim Biochemicals, 1992). This is schematically shown inFIG. 1.

The 293 cells (passage 35, 60% confluency) were inoculated 24 hoursprior to the transfecti on in either 60 mm dishes or 24-well plates. Thecells in each well were tranfected with: 30 μl DOTAP, 2 μg of p53expression vector, and 3 μg of plasmid pJM17. After transfection cellswere fed with the MEM medium every 2-3 days until the onset of CPE.

EXAMPLE 3 Confirming the Identity of Recombinant Adenovirus

This example illustrates a new polymerase chain reaction (PCR) assay forconfirming the identity of recombinant virions following cotransfectionof the appropriate cell line.

Aliquots of cell culture supernatants (50 to 370 μl) were collected fromthe test plates, treated with proteinase K (50 μg/ml with 0.5% SDS and20 mM EDTA) at 56° C. for 1 hour, extracted with phenol-chloroform, andthe nucleic acids were ethanol precipitated. The DNA pellets wereresuspended in 20 μl dH₂O and used as template for PCR amplification.The relative locations of the PCR primers and their sequences aredepicted in FIG. 1 and are SEQ ID NOS:1, 2, 3 and 4, respectively. ThecDNA insert-specific primers define a 1.4 kb PCR product and the viralgenome-specific primers define a 0.86 kb PCR product. The PCR reactionswere carried out in a 50 μl volume containing 4 mM MgCl₂, 50 mM KCl,0.1% triton X-100, 200 μM each of dNTPs, 10 mM Tris-Ci (pH 9.0), 2 μM ofeach primer, and 1.0 unit of Taq polymerase (Promega). The reactionswere carried out at 94° C., 0.5 min, 56° C., 0.5 min, and 72° C., 1 minfor 30 cycles.

In order to simplify the procedure of identification of newly propagatedrecombinant virus, a direct PCR assay on DNA samples from cell culturesupernatant was developed. Aliquots (50 or 370 μl) of the cell mediumsupernatant with CPE were treated with proteinase K andphenol/chloroform extraction. After ethanol precipitation, the DNAsamples were analyzed using PCR employing two pairs of primers toamplify insert-specific and viral-genome-specific sequences. The PCRprimer targets and their sequences are depicted in FIG. 1. Primers 1, 2,3 and 4 are represented by SEQ ID NOS:1, 2, 3 and 4, respectively.

As a result, a 1.4 kb cDNA insert and a 0.86 kb viral genome fragmentwere amplified from the expression vector (positive control) and the DNAsamples of the positive cell culture (FIG. 2B, lane 1 and 4,respectively). Only the 0.86 kb fragment was amplified from the DNAsample of Ad5/RSV/GL2 virus (negative control, lane 2). No amplifiedbands appeared from PCR reactions that used either untreated positivecell culture medium supernatant (lane 3).

These results indicated that adenoviruses released into cell culturemedium are detectable by PCR, using as little as 50 μL of the cellculture medium supernatant for preparing DNA templates. These resultswill allow development of a quantitative method for using this techniqueto determine adenovirus titers, traditionally done by plaque assays.

The wild-type sequence of the p53 cDNA in the Ad5CMV-p53 virus wasconfirmed by dideoxy DNA sequencing on the CsCl-gradient-purified viralDNA. The control virus Ad5/RSV/GL2, generated in a similar manner, has astructure similar to that of Ad5CMV-p53 except a Rous sarcoma viralpromoter and luciferase cDNA were used in its expression cassette. Therecombinant adenovirus that carries a E. coli b-galactosidase gene(LacZ), Ad5CMV-LacZ, also has a structure similar to that of Ad5CMV-p53,and was obtained from Dr. Frank L. Graham.

Viral stock, titer, and infection. Individual clones of the Ad5CMV-p53,Ad5/RSV/GL2, and Ad5CMV-LacZ viruses were obtained byplaque-purification according to the method of Graham and Prevec (1991).Single viral clones were propagated in 293 cells. The culture medium ofthe 293 cells showing the completed cytopathic effect was collected andcentrifuged at 1000× g for 10 min. The pooled supernatants werealiquoted and stored at −20° C. as viral stocks. The viral titers weredetermined by plaque assays (Graham and Prevec, 1991). Infections of thecell lines were carried out by addition of the viral solutions (0.5 mlper 60-mm dish) to cell monolayers and incubation at room temperaturefor 30 min with brief agitation every 5 min. This was followed by theaddition of culture medium and the return of the infected cells to the37° C. incubator.

The gene transfer efficiency of the recombinant adenoviruses was alsoevaluated using Ad5CMV-LacZ in a variety of cell lines such as H226Br,H322, H460, HeLa, Hep G2, LM2, and Vero. By X-gal staining, all of thecell lines were stained 97-100% blue after infection with Ad5CMV-LacZ atan MOI of 30 PFU/cell.

EXAMPLE 4 Ad5CMV-p53-Directed p53 Gene Expression in Human Lung CancerCells

This example describes the use of recombinant p53 adenovirus to infecthuman lung cancer cells with a homozygous p53 gene deletion. The resultsshow that growth of these cells and expression of mutant p53 wassuppressed, indicating the potential of the Ad5CMV-p53 virion as auseful agent for control of metastatic cells.

Immunohistochemistry was performed on infected cell monolayers that werefixed with 3.8% formalin and treated with 3% H202 in methanol for 5 min.Immunohistochemical analysis was performed using Vectastain Elite kit(Vector, Burlingame, Calif.). The primary antibody used was anti-p53antibody PAb 1801 (Oncogene Science, Manhasset, N.Y.); MOPC-21 (OrganonTeknika Corp., West Chester, Pa.) was used as a negative control. Thesecond antibody was an avidin-labeled anti-mouse IgG (Vector). Thebiotinylated horseradish peroxidase ABC complex reagent was used todetect the antigen-antibody complex. Finally the cells werecounterstained with Harris hematoxylin (Sigma) and mounted with Cytoseal60 (Stephens Scientific, Riverdale, N.J.).

Immunohistochemical analysis of the infected cell lines was performed toexamine the in situ expression of p53 expression driven by the CMVpromoter of the Ad5CMV-53 virus. In the H358 cell line, which has ahomozygous deletion of p53, the p53 gene was transferred with 97-100%efficiency, as detected by immunohistochemical analysis, when the cellswere infected with Ad5CMV-p53 at a multiplicity of infection of 30-50plaque-forming units (PFU)/cell (FIG. 4).

The high transfer efficiency of recombinant adenovirus was confirmed byAd5CMV-LacZ, a virus which carries the LacZ gene transcribed by thehuman CMV IE promoter. At an MOI of 30-50 PFU/cell, all of the cellsexamined, including HeLa, Hep G2, LM2, and the human NSCLC cancer celllines were 97-100% positive for b-galactosidase activity by X-galstaining. These results indicate that adenoviral vectors are anefficient vehicle for gene transfer into human cancer cells.

Western blotting analysis was performed on total cell lysates preparedby lysing monolayer cells in dishes with SDS-PAGE sample buffer (0.5 mlper 60-mm dish) after rinsing the cells with phosphate-buffered saline(PBS). For SDS-PAGE analysis lanes were loaded with cell lysatesequivalent to 5×10⁴ cells (10-15 ml). The proteins in the gel weretransferred to Hybond™-ECL membrane (Amersham, Arlington Heights, Ill.).The membranes were blocked with 0.5% dry milk in PBS and probed with theprimary antibodies: mouse anti-human p53 monoclonal antibody PAb 1801and mouse anti-human β-actin monoclonal antibody (Amersham), washed andprobed with the secondary antibody: horseradish peroxidase-conjugatedrabbit anti-mouse IgG (Pierce Chemical Co., Rockford, Ill.). Themembranes were developed according to the Amersham's enhancedchemiluminescence protocol. Relative quantities of the exogenous p53expressed were determined by densitometer (Molecular Dynamics Inc.,Sunnyvale, Calif.).

Western blots showed the exogenous p53 protein was expressed at a highlevel (FIG. 5A lanes 2,3 and 5,6). The protein peaked at day 3 afterinfection (FIG. 6, insert, 0.5 days to 3 days). As a control, a virionwith a structure similar to the recombinant Ad5CMV-p53 of Example 1 wasconstructed. This virion contains a luciferase cDNA driven by RousSarcoma Virus LTR promoter in the expression cassette of the virion.Neither p53 expression nor change in actin expression was detected inthe cells infected by the virion Ad5/RSV/GL2.

The recombinant p53 adenovirus was used to infect three human lungsNSCLC cell lines: cell line H358, which has a homozygous deletion of thep53 gene, cell line H322, which has a point mutation of the p53 gene atcodon 248 (G to T), and cell line H460, which has a wild-type p53 gene.The growth rate of human NSCLC cells was determined following theinoculation of H322 and H460 (1×10⁵) or H358 (2×10⁵) in 60-mm culturedishes 24 h before viral infection. The cells were infected with theviruses at a multiplicity of infection (MOI) of 10 PFU/cell. Culturemedium was used for the mock infection control. Triplet cultures of eachcell line with different treatments were counted daily for days 1-6after infection.

Growth of the H358 cells infected with Ad5CMV-p53 was greatly inhibitedin contrast to that of noninfected cells or the cells infected with thecontrol virion (FIG. 7A). Growth of H322 cells was also greatlyinhibited by the p53 virion (FIG. 7B), while that of human lung cancerH460 cells containing wild type p53 was affected to a lesser degree(FIG. 7C). Growth of the Ad5CMV-p53 virus-infected H358 cells wasinhibited 79%, whereas that of noninfected cells or the cells infectedwith the control virus were not inhibited. Growth of cell line H322,which has a point mutation in p53, was inhibited 72% by Ad5CMV-p53,while that of cell line H460 containing wild-type p53 was less affected(28% inhibition).

The results indicate that the p53 recombinant adenovirus possessesproperties of tumor suppression, working through restoration of the p53protein function in tumor cells.

EXAMPLE 5 Ad5CMV-p53 in the Treatment of p53 Deficient Cells

The present example concerns the use of recombinant p53 adenovirus torestore growth suppression of tumor cells in vitro and thus to treat themalignant or metastatic growth of cells. It describes some of the waysin which the present invention is envisioned to be of use in thetreatment of cancer via adenovirus-mediated gene therapy.

H358 cells were infected with Ad5CMV-p53 and Ad5/RSV/GL2 at a MOI of 10PFU/cell. An equal amount of cells were treated with medium as a mockinfection. Twenty-four hours after infection, the treated cells wereharvested and rinsed twice with PBS. For each treatment, three million(3×10⁶) cells in a volume of 0.1 ml were injected s.c. to each nudemouse (Harlan Co., Houston, Tex.). Five mice were used for eachtreatment. Mice were irradiated (300 cGy, ⁶⁰Co) before injection andexamined weekly after injection. Tumor formation was evaluated at theend of a 6-week period and tumor volume was calculated by assuming aspherical shape with the average tumor diameter calculated as the squareroot of the product of cross-sectional diameters.

To determine the inhibitory effect on tumorigenicity mediated byAd5CMV-p53 nude mice were injected s.c. with H358 cells (a humanNSCLC-type cell) to induce neoplastic growth. Each mouse received oneinjection of cells that had been infected with Ad5CMV-p53 or Ad5/RSV/GL2at 10 PFU/cell for 24 h. H358 cells treated with medium alone were usedas mock-infected controls. Tumors, first palpable at postinjection day14, were induced only by the mock- or control virus-infected cells asdemonstrated in Table I:

TABLE I Effect of Ad5CMV-p53 on tumorigenicity of H358 in nude mice^(a)No. of Tumors/ Mean Volume Treatment No. of Mice (%) (mm³ ± SD) Medium4/5 (80) 37 ± 12 Ad5/RSV/GL2 3/4 (75) 30 ± 14 Ad5CMV-p53 0/4 (0)  —^(a)The treated H358 cells were injected s.c. at 2 × 10⁶ cells/mouse.Tumor sizes were determined at the end of a 6-week period.

As shown in Table 1 mice that received Ad5CMV-p53-treated cells did notdevelop tumors. The tumors at the end of a 6-week period were 4-10 mm indiameter. This study was initiated with five mice per group; one mouseeach in the Ad5CMV-p53 or Ad5/RSV/GL2 group failed to complete thestudy. The early deaths were presumably due to nosocomial infection.

EXAMPLE 6 Ad5CMV-p53 in the Treatment of Lung Cancer

The present example concerns the use of recombinant p53 adenovirus torestore growth suppression of tumor cells in vivo and thus to treatcancers in animals. It describes some of the ways in which the presentinvention is envisioned to be of use in the treatment of cancer viaadenovirus-mediated gene therapy.

The efficacy of Ad5CMV-p53 in inhibiting tumorigenicity was furtherevaluated in the mouse model of orthotopic human lung cancer. Since H358and H322 cells did not produce tumors in this model, cell line H226Brwas used. This cell line has a squamous lung cancer origin andmetastasized from lung to brain. H226br has a point mutation (ATC toGTC) at exon 7, codon 254, of the p53 gene and is tumorigenic in mice.

The procedure for tests in the mouse model of orthotopic human lungcancer has been previously described (Georges, et al., 1993). Briefly,nude mice treated with radiation (300 cGy, ⁶⁰Co) were inoculated withH226Br cells by intratracheal instillation. Each mouse received 2×10⁶cells in a volume of 0.1 ml PBS. Three days after inoculation, 10 miceper group were treated with 0.1 ml of viruses or vehicle (PBS) byintratracheal instillation once a day for two days. The virus dosageused was 5×10⁷ Ad5CMV-p53 or Ad5/RSV/GL2 per mouse. The mice wereeuthanized at the end of a 6-week period. Tumor formation was evaluatedby dissecting the lung and mediastinum tissues and measuring the tumorsize. The tumors were confirmed by histologic analysis of the sectionsof the tumor mass.

The irradiated nude mice were inoculated with 2×10⁶H226Br cells/mouse byintratracheal instillation. Three days after inoculation, each of themice (8-10 mice per group) were treated with 0.1 ml of either Ad5CMV-p53or Ad5/RSV/GL2 or vehicle (PBS) by intratracheal instillation once a dayfor two days. The virus dosage used was 5×10⁷ PFU/mouse. Tumor formationwas evaluated at the end of a 6-week period by dissecting the lung andmediastinum tissues and measuring the tumor size. A flow chart of theprocedure is depicted in FIG. 7, with representative samples ofdissection demonstrated in FIG. 8. The detected tumors were confirmed byhistologic analysis. The data of tumor measurements are summarized inTable II:

TABLE II Effect of Ad5CMV-p53 on tumorigenicity of H226Br in mouse modelof orthotopic human lung cancer^(a) No. mice with Tumors/ Mean VolumeTreatment Total Mice (%) (mm³ ± SD) Vehicle 7/10 (70) 30 ± 8.4Ad5/RSV/GL2 8/10 (80) 25 ± 6.9 Ad5CMV-p53 2/8 (25)    8 ± 3.3^(b)^(a)Mice were inoculated with 2 × 10⁶ H226Br cells/mouseintratracheally. On the 3rd day postinoculation, the mice were giveneither vehicle or viruses (5 × 10⁷ each in 0.1 ml) intratracheally oncea day for 2 days. Tumor formation was evaluated at the end of a 6-weekperiod. ^(b)p < 0.05 two-way analysis of variance when compared to thegroups receiving vehicle (PBS) or virus control.

Only 25% of the Ad5CMV-p53-treated mice formed tumors, whereas in thevehicle or Ad5/RSV/GL2 control group, 70-80% of the treated mice formedtumors. The average tumor size of the Ad5CMV-p53 group was significantlysmaller than those of the control groups. These results indicate thatAd5CMV-p53 can prevent H226Br from forming tumors in the mouse model oforthotopic human lung cancer.

EXAMPLE 7 Ad5CMV-p53 in Treatment Regimens

Naturally, animal models will be employed as part of the pre-clinicaltrials, as described herein in Examples 5 and 6. Thereafter, patientsfor whom the medical indication for adenovirus-mediated gene transfertreatment has been established may be tested for the presence ofantibodies directed against adenovirus. If antibodies are present andthe patient has a history of allergy to either pharmacological ornaturally occurring substances, application of a test dose of on theorder of 10³ to 10⁶ recombinant adenovirus under close clinicalobservation would be indicated.

For the treatment of cancer using Ad5CMV-p53, recombinant adenovirusexpressing p53 under the control of suitable promoter/enhancer elements,such as the CMV promoter, would be prepared and purified according to amethod that would be acceptable to the Food and Drug Administration(FDA) for administration to human subjects. Such methods include, butare not limited to, cesium chloride density gradient centrifugation,followed by testing for efficacy and purity.

Two basic methods are considered to be suitable for p53 adenovirustreatment methods, a direct or local administration and a more generaladministration. The present methods are suitable for treating any of thevariety of different cancers known to be connected with p53 mutations.In regard to general administration, a simple intravenous injection ofadenovirus has been shown to be sufficient to result in viral infectionof tissues at sites distant from the injection (Stratford-Perricaudet etal., 1991b), and is thus suitable for the treatment of all p53-linkedmalignancies. The virus may be administered to patients by means ofintravenous administration in any pharmacologically acceptable solution,or as an infusion over a period of time. Generally speaking, it isbelieved that the effective number of functional virus particles to beadministered would range from 1×10¹⁰ to 5×10¹².

Also, particularly where lung cancer is concerned, more direct physicaltargeting of the recombinant adenovirus could be employed if desired, inan analogous manner to the intratracheal administration of the cysticfibrosis transmembrane conductance regulator (Rosenfeld et al., 1992).This would result in the delivery of recombinant p53 adenovirus closerto the site of the target cells.

In more detail, preferred treatment protocols may be developed along thefollowing lines. Patients may first undergo bronchoscopy to assess thedegree of obstruction. As much gross tumor as possible should beresected endoscopically. Patients should preferably undergo bronchoscopyunder topical or general anesthesia. A Stifcor™ transbronchialaspiration needle (21g) will be passed through the biopsy channel of thebronchoscope. The residual tumor site would then be injected with thep53 adenovirus in a small volume such as about 10 ml or less.

In any event, since the adenovirus employed will be replicationincompetent, no deleterious effect of the virus itself on subject healthis anticipated. However, patients would remain hospitalized during thetreatment for at least 48 hours to monitor acute and delayed adversereactions. Safety-related concerns of the use of replication deficientadenovirus as a gene transfer vehicle in humans have been addressed inthe past (Rosenfeld et al., 1992; Jaffe et al., 1992), but the dose ofadenovirus to be administered should be appropriately monitored so as tofurther minimize the chance of untoward side effects.

There are various criteria that one should consider as presenting theexistence of a need for response or the existence of toxicity. To assistin determining the existence of toxicity, the tumor bed should bephotographed prior to a course of therapy. The longest diameter and itsperpendicular will be measured. Size will be reported as the product ofthe diameters. From these data, one can calculate from these numbers therate of regrowth of the tumor.

The time to progression can also be measured from the first observationwith reduction in tumor bulk until there is evidence of progressivedisease. Progressive Disease is defined as an increase of ≧25% in thesum of the products of the diameters of the measured lesion. Patientsmust have received at least two courses of therapy before a designationof progression is made. The survival of patients will be measured fromentry into protocol.

Follow-up examinations would include all those routinely employed incancer therapy, including monitoring clinical signs and taking biopsiesfor standard and molecular biological analysis in which the pattern ofexpression of various p53 genes could be assessed. This would alsosupply information about the number of cells that have taken up thetransferred gene and about the relative promoter strength in vivo. Basedon the data obtained adjustments to the treatment may be desirable.These adjustments might include adenovirus constructs that use differentpromoters or a change in the number of pfu injected to ensure ainfection of more, or all, tumor cells without unphysiologicaloverexpression of the recombinant genes.

It is contemplated that the expression of exogenous genes transferred invivo by adenovirus can persist for extended periods of time.Therapeutically effective long-term expression of virally transferredexogenous genes will have to be addressed on a case by case basis.Marker genes are limited in their usefulness to assess therapeuticallyrelevant persistence of gene expression as the expression levelsrequired for the amelioration of any given genetic disorder might differconsiderably from the level required to completely cure another disease.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims. All claimedmatter and methods can be made and executed without undueexperimentation.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Bargonetti, et al. (1991) Cell 65:1083-1091.

Boeheringer Mannheim Biochemicals (1992). DOTAP for high efficiencytransfections, BMBiochemica 9(1):17.

Boshart, M. et al. (1985). A very strong enhancer is located upstream ofan immediate early gene of human cytomegalovirus. Cell, 41:521-530.

Bishop (1987) Science 235:305-311.

Casey, G. Lo-Hueh, M., Lopez, M. E., Vogelstein, B., and Stanbridge, E.J. (1991). Growth suppression of human breast cancer cells by theintroduction of a wild-type p53 gene. Oncogene 6:1791-1797.

Dai, et al. (1992) Proc. Natl. Acad. Sci. 89:10892-10895.

Fields et al. (1990) Science 249:1046-1049.

Georges et al. (1993) Cancer Res 53:1743-1746.

Ghosh-Choudhury and Graham (1982) Biochem. Biophys. Res. Comm.147:964-973.

Gluzman et al., (1982) in Eukaryotic Viral Vectors (Gluzman, Y., Ed.)pp. 187-192, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

Graham, F. L. and A. J. van der Eb. (1973). A new technique for theassay of infectivity of human adenovirus 5 DNA. Virology 52:456-467.

Graham, F. L., J. Smiley, W. C. Russell and R. Nairn (1977).Characteristics of a human cell line transformed by DNA from humanadenovirus type 5. J. Gen Wrol. 36:59-72.

Grunhaus, A. and Horwitx, M. S. (1992). Adenoviruses as cloning vectors.Semin. Virology 3:237-2542.

Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. (1991). p53mutations in human cancers. Science 253:49-53.

Jaffe et al., (1992) Nature Genetics 1:372-378.

Le Gal et al., (1993) Science 259:988-990.

McGrory, W. J. et al. (1988). A simple technique for the rescue of earlyregion I mutations into infectious human adenovirus type 5. Virology163:614-617.

Mercer, W. E. (1992). Cell cycle regulation and the p53 tumor suppressorprotein. Critic. Rev. Eukar. Gene Express. 2:251-263.

Mietz, et al. (1992) EMBO 11:5013-5020.

Montenarh, M. (1992). Biochemical, immunological, and functional aspectsof the growth-suppressor/oncoprotein p53. Critic. Rev. Onco. 3:233-256.

Mulligan, (1993), Science 260:926.

Ragot et al., (1993) Nature, 361:647-650.

Rosenfeld et al., (1991) Science, 232:431434.

Rosenfeld et al., (1992) Cell 68:143-155.

Shaw, et al., (1992) 89:44954499.

Spandidos, et al. (1989), J. Pathol., 157:1-10.

Stratford-Perricaudet, L. and M. Perricaudet. (1991a). Gene transferinto animals: the promise of adenovirus. p. 51-61, In O.Cohen-Haguenauer and M. Boiron (Eds.), Human Gene Transfer, EditionsJohn Libbey Eurotext, France.

Stratford-Perricaudet et al., (1991b) Hum. Gene. Ther. 1:241-256

Takahashi, T., Carbone, D., Takahashi, T., Nau, M. M., Hida, T.,Linnoila, I., Ueda, R., and Minna, J. D. (1992). Wild-type but notmutant p53 suppresses the growth of human lung cancer cells bearingmultiple genetic lesions. 1992. Cancer Res. 52:2340-2342.

Tooza, J. (1981). Molecular biology of DNA Tumor viruses, 2nd ed. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

Travali, et al. (1990), FASEB, 4:3209-3214.

Yonish, et al. (1991), Nature 352:345-347.

Weinberg, R. A. (1991). Tumor suppressor gene. Science 254:1138-1145.

Wilcock, et al. (1991) Nature 349:429431.

Zakut-Houri et al. (1985), EMBO J., 4:1251-1255.

Zhang, et al. (1993) BioTechniques in press.

4 19 Nucleic acid Single Linear <Unknown> 1 GGCCCACCCC CTTGGCTTC 19 20Nucleic acid Single Linear <Unknown> 2 TTGTAACCAT TATAAGCTGC 20 20Nucleic acid Single Linear <Unknown> 3 TCGTTTCTCA GCAGCTGTTG 20 20Nucleic acid Single Linear <Unknown> 4 CATCTGAACT CAAAGCGTGG 20

What is claimed is:
 1. A recombinant adenovirus that includes anexpression construct comprising a sequence encoding p53 under thecontrol of a promoter.
 2. The recombinant adenovirus of claim 1, whereinthe expression construct further comprises a polyadenylation signal. 3.The recombinant adenovirus of claim 1, wherein the recombinantadenovirus is replication deficient.
 4. The recombinant adenovirus ofclaim 1, wherein the promoter is a cytomegalovirus IE.
 5. Therecombinant adenovirus of claim 3, wherein the expression constructlacks the E1A and E1B regions.
 6. The recombinant adenovirus of claim 3,wherein the p53-encoding sequence replaces the E1A and E1B regions ofthe expression construct.
 7. The recombinant adenovirus of claim 5,wherein the expression construct lacks the E3 region.
 8. The recombinantadenovirus of claim 5, wherein the expression construct lacks the E2region.
 9. The recombinant adenovirus of claim 5, wherein the expressionconstruct lacks the E4 region.
 10. The recombinant adenovirus of claim6, wherein the adenovirus has the genome structure of FIG.
 1. 11. Apharmaceutical composition comprising: (a) a recombinant adenovirus thatincludes an expression construct comprising a sequence encoding p53under the control of a promoter; and (b) a pharmaceutically acceptablecarrier, excipient or diluent.
 12. The pharmaceutical composition ofclaim 11, wherein the expression construct further comprises apolyadenylation signal.
 13. The pharmaceutical composition of claim 11,wherein the promoter is a cytomegalovirus immediate early promoter. 14.The pharmaceutical composition of claim 12, wherein the recombinantadenovirus is replication deficient.
 15. The pharmaceutical compositionof claim 14, wherein the expression construct lacks the E1A and E1Bregions.
 16. The pharmaceutical composition of claim 15, wherein thep53-encoding sequence replaces the E1A and E1B regions of the expressionconstruct.
 17. The pharmaceutical composition of claim 16, wherein theadenovirus has the genome structure of FIG.
 1. 18. The pharmaceuticalcomposition of claim 17, wherein the expression construct lacks the E3region.
 19. The pharmaceutical composition of claim 17, wherein theexpression construct lacks the E2 region.
 20. The pharmaceuticalcomposition of claim 17, wherein the expression construct lacks the E4region.
 21. A recombinant host cell that includes an adenoviralexpression construct comprising a sequence encoding p53 under thecontrol of a promoter.
 22. The recombinant host cell of claim 21,wherein the promoter is a cytomegalovirus immediate early promoter. 23.The recombinant host cell of claim 21, further comprising a nucleic acidencoding the E1A and E1B regions of adenovirus.
 24. The recombinant hostcell of claim 21, further comprising a nucleic acid encoding the E2region of adenovirus.
 25. The recombinant host cell of claim 21, furthercomprising a nucleic acid encoding the E4 region of adenovirus.